Metabolic Reprogramming of Acute lymphoblastic Leukemia Cells in Response to Glucocorticoid Treatment

Introduction

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Standard treatment involves glucocorticoids (GCs) like prednisolone and dexamethasone (dex), which are highly effective but can lead to relapse and long-term adverse effects. GC sensitivity predicts treatment outcome, yet the mechanisms of GC-induced cell death and resistance remain unclear. While GC-induced apoptosis is mediated by the GC receptor (NR3C1), resistance mechanisms are complex. Previous studies have identified GC-regulated genes and expression patterns predictive of sensitivity, but the underlying molecular mechanisms are not fully understood. GCs are known metabolic regulators, generally opposing insulin action and inducing catabolic states. In immune cells, GCs suppress inflammation and inhibit immune responses. However, specific metabolic reprogramming in ALL cells by GCs and its role in cell death are less understood. While some studies show altered metabolic gene expression, direct metabolite and isotope-tracing data are scarce. GC treatment causes autophagosome accumulation in ALL cells, suggesting a catabolic state. GCs suppress glucose uptake, potentially via GLUT1 inhibition, but whether this causes cell death or is a consequence of it is unclear. Reducing glucose can sensitize cells to GCs, but GC-induced apoptosis is ATP-dependent, and ATP loss typically leads to necrosis, not apoptosis. Blocking GC-induced autophagy can prevent cell death, indicating autophagy is detrimental to ALL cells and GC-induced death is not solely due to nutrient energy crisis. This study aims to elucidate the metabolic reprogramming caused by GCs in ALL cells.

Results

Response of RS4;11 cells to dexamethasone

RS4;11 cells, a GC-sensitive pre-B ALL line, showed a decline in viable cell numbers to approximately one-third after 36 hours of dexamethasone (dex) treatment (Fig. 1a). The ADP/ATP ratio increased after 12 hours, indicating decreased adenylate charge, and AMP-activated protein kinase (AMPK) phosphorylation was observed at 24 hours (Fig. 1b, c). Autophagosome marker LC3-II accumulated between 8 and 16 hours, while p62, a protein degraded by autophagy, decreased at 24 hours. Inhibition of lysosomal degradation with bafilomycin A1 (BafA1) further increased LC3-II and blocked p62 degradation, confirming autophagic flux (Fig. 1d, e). Late apoptotic cells (Annexin V+/PI+) increased significantly between 24 and 48 hours, indicating that autophagy precedes apoptosis, which continues to increase over time. Thus, GC-treated RS4;11 cells undergo ATP depletion, autophagy induction, and eventually apoptotic cell death.

Time-course analysis of the proteome

A high-coverage proteomics analysis of RS4;11 cells treated with dex revealed gradual changes in protein expression, with most pronounced differences at 24 hours, coinciding with apoptosis onset (Fig. 2a, b). Known GC-responsive proteins like TSC22D3 were induced, while cell cycle progression proteins like CDK4 decreased. Antiproliferative proteins such as TGF-beta (TGFB1) increased, consistent with cell cycle arrest. Apoptotic markers BCL2L11 and CD93 emerged at 16–24 hours (Fig. 2c-f). The proto-oncogene c-myc (MYC), known to be downregulated by GCs, decreased early (4 hours) and its target genes were significantly downregulated, suggesting MYC suppression contributes to reduced glycolysis by GCs.

Metabolic events in GC-treated RS4;11 cells

To investigate metabolic effects, cells were traced with 1-13C-glucose, U-13C, 15N-glutamine, 3-13C-serine, and U-13C-methionine. GC treatment rapidly decreased pyrimidine synthesis intermediates (orotate, dihydroorotate) and prevented 13C labeling, indicating de novo pyrimidine synthesis inhibition. mRNA for DHODH, a pyrimidine synthesis enzyme, also declined. Thymidylate synthase was downregulated. 13C labeling of purines and purine synthesis enzymes also declined. Conversely, purine nucleobases (hypoxanthine, guanine) accumulated. Putrescine, the first metabolite of polyamine synthesis, decreased, along with reduced mRNA for ODC1 and AMD1, key enzymes in polyamine synthesis. This suggests suppressed cell proliferation and induced apoptosis. Fatty acid synthesis enzymes were downregulated, while fatty acid oxidation enzymes were upregulated. CDP-choline, crucial for phospholipid synthesis, increased, suggesting its role in membrane synthesis for apoptotic processes.

GCs alter fuel usage and promote glutamine synthesis

GCs typically inhibit glucose uptake and utilization. In RS4;11 cells, glucose transporters SLC2A1 and SLC2A3 mRNA and protein levels were repressed, and lactate accumulation was reduced, consistent with suppressed glycolysis (Fig. 4a, b, c). The contribution of glucose-derived pyruvate to the TCA cycle diminished, indicated by reduced 13C labeling of TCA cycle metabolites like fumarate and aconitate (Fig. 4d-g). This might explain the declining energy charge and AMPK activation. While inhibiting glycolysis with 2-deoxyglucose did not induce autophagy or cell death to the same extent as dex treatment, other mechanisms are important. Essential amino acid uptake was reduced. Glutamine catabolism can be induced when glucose metabolism is inhibited, but here, glutamine uptake did not increase; instead, total glutamine abundance increased, suggesting increased glutamine synthesis. The glutamine-synthesizing enzyme glutamate-ammonia ligase (GLUL) was strongly induced at both mRNA and protein levels by dex treatment (Fig. 4i, j, k). This induction of GLUL was observed in GC-sensitive but not GC-resistant cells. Glutaminase (GLS) and glutamate dehydrogenase (GLUD) levels were not altered. GLUL induction by corticoids has been reported in leukemia and normal tissues, but its function was unclear.

Role of glutamine metabolism in dex-induced autophagy and cell death

Experiments investigated the role of glutamine metabolism in GC-induced autophagy and cell death. Removing glutamine from the medium inhibited autophagic flux and reduced cleaved caspase 3 in dex-treated cells, indicating glutamine presence modulates autophagy. Ammonium (NH4+), generated from protein catabolism and utilized by GLUL, can inhibit lysosomal degradation and induce autophagy. Supplementing medium with NH4Cl caused LC3-II accumulation, comparable to dex treatment. Glutamine-containing medium showed NH4+ production by ALL cells, with glutamine accounting for most of it. Adding dimethyl-a-ketoglutarate (dm-akg), which converts to a-kg, almost completely prevented dex-induced LC3-II accumulation and caspase 3 cleavage. Removing glutamine had similar effects. dm-akg supplementation increased glutamate production and reduced glutamine uptake, consistent with increased NH4+ assimilation by GLUL. This suggests NH4+ scavenging by GLUL is integral to dex-induced catabolism, modulating autophagy and cell death. A schematic illustrates this proposed mechanism (Fig. 5i).

Discussion

Understanding GC mechanisms is crucial for overcoming resistance and developing alternative therapies. This study detailed the metabolic reprogramming of ALL cells by GCs using isotope tracing, mass spectrometry, and proteomics. Findings recapitulate known GC effects and show metabolic events consistent with growth arrest and apoptosis, including suppressed nucleotide and polyamine synthesis. A coordinated program of suppressed fatty acid/sterol synthesis and increased fatty acid oxidation was observed, potentially linked to autophagy induction. CDP-choline synthesis was induced, suggesting phospholipid synthesis is required for apoptotic processes like membrane blebbing. The study acknowledges limitations: isotope tracing data is suggestive due to simultaneous metabolic flux changes; attributing specific metabolic events to growth arrest versus apoptosis is challenging without further experiments; and in vivo conditions (e.g., hypoxia) may differ. The marked induction of GLUL gene was observed, consistent with its role in capturing excess NH4+ from amino acids in other tissues. In ALL cells, GCs induce autophagy, reduce amino acid uptake, and suppress glucose/glutamine entry into the TCA cycle, leading to a catabolic phenotype. This supports investigating GLUL as an ammonia scavenger involved in autophagy and cell death. While the role of autophagy in cell death is debated (cytoprotective vs. detrimental), this study's findings in B-ALL cells align with previous work showing detrimental autophagy. Low GLUL expression predicts poor outcome in ALL, supporting its integral role in GC response. Further examination of GLUL is warranted to understand its role in autophagy and GC-induced ALL cell death.

Materials and Methods

Two pre-B ALL cell lines, RS4;11 and SupB-15, were cultured in RPMI 1640 medium supplemented with HEPES, heat-inactivated fetal calf serum, L-glutamine, streptomycin, and penicillin. Cells were treated with dexamethasone (dex) at various concentrations. For ammonia measurements, an Ammonia assay kit was used. ADP/ATP ratio was determined using an assay kit. Metabolomics and isotope tracing involved a custom RPMI medium with isotopically labeled glucose, glutamine, serine, and methionine. Metabolites were extracted and analyzed using Thermo QExactive orbitrap coupled to HILIC chromatography. Quantitative 13C standards were used for medium metabolite quantification via LC-HRMS. Viability, apoptosis, and autophagy were assessed using CellTiterBlue, WST-1, Annexin V/PI staining with FACS, and western blotting for LC3B and p62. RNA was extracted and analyzed by qRT-PCR using KAPA 2 G SYBRGreen. Proteomics involved cell lysis, protein digestion (modified FASP protocol), TMT10plex labeling, peptide fractionation via IPG-IEF, and LC-MS/MS analysis on Q Exactive or Q Exactive HF instruments. Statistical analysis included hierarchical clustering and GSEA-p method.